Variant forms ofα-2-l-fucosyltransferase in human submaxillary glands from blood group ABH “secretor” and “non-secretor” individuals

The properties of the -2-l-fucosyltransferases in submaxillary gland preparations from blood group ABH secrefors and non-secretors were compared. The level of activity in the non-secretor gland homogenates amounted to about 5% only of that found in the secretor gland preparations. The enzymes from the two sources differed in solubility properties, charge and affinities for donor and acceptor substrates. The enzyme from secretor glands showed a preference for acceptors with Type 1 [d-galactosyl(1–3)-N-acetyl-d-glucosamine] structures whereas the enzyme from non-secretor glands had a preference for Type 2 [d-galactosyl(1–4)-N-acetyl-d-glucosamine] structures.These results demonstrate that expression of the secretor gene (Se) is associated with a molecular form of the -2-l-fucosyltransferase that is different from the species present in the same tissue when theSe gene is not expressed.     

Micropropagation of flowering dogwood (Cornus florida) from seedlings

A method for regenerating whole plants from nodal (axillary bud) cultures of seedlings was developed for flowering dogwood (Cornus florida L.). The seed source significantly influenced the rate of proliferation, although cultures initiated from each of the seven mother trees produced some shoots. Woody plant medium (WPM) was superior to either Murashige and Skoog or Schenk and Hildebrandt basal medium. 6-Benzyladenine (BA) at 2.2 or 4.4 m stimulated the generation of significantly more useable shoots (1 cm) compared to all other concentrations (0.5–22.5 m tested. Thidiazuron (TDZ) at 0.6 and 1.1 m supported proliferation, but strongly inhibited shoot elongation. TDZ initiated cultures transferred to medium containing 4.4 m BA produced usable shoots after five additional subcultures. Shoots generated adventitious roots when exposed to either a 12-h pulse of relatively high concentrations (246–1230 m or continuous lower concentrations (0.5–49.0 m of indolebutyric acid (IBA) for longer periods. Microshoots produced the significantly greatest number of roots when subjected to 4.9 m IBA in WPM over a 4-week period. Whole plants were acclimatized to the laboratory conditions and subsequently to the greenhouse. The methodology described here should be useful in a breeding program by supplying multiple copies of unique, recombinant genotypes.     

The microbial production of 3aα-H-4α- (3′-propionic acid)-5α-hydroxy-7aβ-methylhexahydro-indan-1-one-δ-lactone from cholesterol

Summary A mutant strain of Rhodococcus australis CSIR-236.457 which accumulates 3a-H-4-(3-propionic acid)-5-hydroxy-7a-methylhexahydro-indan-1-one--lactone from cholesterol, stigmasterol and -sitosterol was studied. The product is produced in a 60% molar yield in a dilute black strap molasses medium containing 6–12g/l cholesterol after a 72 hour fermentation period.     

Regulation of biosynthesis of aspartate family amino acids in the photosynthetic bacterium Rhodopseudomonas palustris

The four amino acids of the aspartate family (l-lysine, l-methionine, l-threonine, and l-isoleucine) are produced in bacteria by a branched biosynthetic pathway. Regulation of synthesis of early common intermediates and of carbon flow through distal branches of the pathway requires operation of a number of subtle feedback controls, which are integrated so as to ensure balanced synthesis of the several end products. Earlier studies with nonsulfur purple photosynthetic bacteria were instrumental in revealing the existence of alternative regulatory schemes, and in this communication we report on the control pattern of a representative of this physiological group not previously investigated, Rhodopseudomonas palustris. The results obtained from study of the properties of four key regulatory enzymes of the aspartate family pathway (-aspartokinase, homoserine dehydrogenase, homoserine kinase, and threonine deaminase) and of the effects of exogenous amino acids (i. e., the end products) on growth of the bacterium indicate that the control schema in Rps. palustris differs substantially from the schemes described for other Rhodopseudomonas species, but resembles the regulatory pattern observed in Rhodospirillum rubrum.Abbreviations A absorbancy - AK -aspartokinase - ASA aspartate -semialdehyde - DTT dithiothreitol - HS l-homoserine - HSDH homoserine dehydrogenase - HSK homoserine kinase - I l-isoleucine - KU Klett-Summerson photometer units - L l-lysine - M l-isoleucine - KU Klett-Summerson photometer units - L l-lysine - M l-methionine - ME -mercaptoethanol - PABA p-aminobenzoic acid - T l-threonine - TD threonine deaminase - RCV synthetic growth medium (see text) - YP agar medium containing 0.3% yeast extract, 0.3% peptone, and 1.5% agar - Y2T synthetic growth medium (see text)     

UDP-GlcNAc: Galβ3GalNAc-Mucin: (GlcNAc → GalNAc) β6-N-Acetylglucosaminyltransferase and UDP-GlcNAc: Galβ3(GlcNAcβ6) GalNAc-Mucin (GlcNAc → Gal)β3-N-Acetylglucosaminyltransferase from Swine Trachea Epithelium

Summary Two specific -N-acetylglucosaminyltransferases involved in the branching and elongation of mucin oligosaccharide chains, namely, a 1,6 N-acetylglucosaminylsaminyltransferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal3GalNAc-Mucin to yield Gal3(GlcNAc6)GalNAc-Mucin and a 3-N-acetylglucosaminyl transferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal3(GlcNAC6)GalNAc-mucin to yield GlcNAc3Gal3 (GlcNAc6)GalNAc-Mucin were purified from the microsomal fraction of swine trachea epithelium. The 1,6-N-acetylglucosaminyltransferase was purified about 21,800-fold by procedures which included affinity chromatography on DEAE columns containing bound asialo Cowper's gland mucin glycoprotein with Gal1,3GalNAc side chains. The apparent molecular weight estimated by gel filtration was found to be about 60 Kd. The purified enzyme showed a high specificity for Gal1,3GalNAc chains and the most active substrates were mucin glycoproteins containing these chains. The apparent Km of the 6-glucosaminyltrans-ferase for Cowper's gland mucin glycoprotein containing Gal1,3GalNAc chains was 0.53 µM; for UDP-N-acetylglucosamine, 12 µM; and for Gal 1,3GalNAc NO₂ø, 4 mM. The activity of the 6-glucosaminyltransferase was dependent on the extent of glycosylation of the Gal3GalNAc chains in Cowper's gland mucin glycoprotein.The best substrate for the partially purified 3-Glucosaminyltransferase was Cowper's gland mucin glycoprotein containing Gal1,3(GlcNAc6)GalNAc side chains. This enzyme showed little or no activity with intact sialylated Cowper's gland mucin glycoprotein or derivatives of this glycoprotein containing GalNAc or Gal1,3GalNAc side chains.The radioactive oligosaccharides formed by these enzymes in large scale reaction mixtures were released from the mucin glycoproteins by treatment with alkaline borohydride, isolated by gel filtration on Bio-Gel P-6 and characterized by methylation analysis and sequential digestion with exoglycosidases. The oligosaccharide products formed by the 6- and 3-glucosaminyltransferases were shown to be Gal3(GlcNAC6) GalNAc and GlcNAc3 Gal3(GlcNAC6)GalNAc respectively.Taken collectively, these results demonstrate that swine trachea epithelium contains two specific N-acetylglucosaminyltransferases which catalyze the initial branching and elongation reactions involved in the synthesis of O-linked oligosaccharide chains in respiratory mucin glycoproteins. The first enzyme a 6-glucosaminyltransferase converts Gal3GalNAc chains in mucin glycoproteins to Gal3(GlcNAc6)GalNAc chains. This product is the substrate for a second 3-glucosaminyltransferase which converts the Gal3(GlcNAc6)GalNAc chains to GlcNAc3Gal(GlcNAc6)GalNAc chains in the glycoprotein. The 3-glucosaminyltransferase did not utilize Gal3GalNAc chains as a substrate and this results in an ordered sequence of addition of N-acetylglucosamine residues to growing oligosaccharide chains in tracheal mucin glycoproteins.Abbreviations NeuNAc N-acetylneuraminic acid - GalNAcol N-acetylgalactosaminitol - CGMG Cowper's gland mucin glycoprotein - GalNAc-CGMG Cowper's gland mucin glycoprotein containing GalNAc side chains O-glycosidically linked to serine or threonine - Gal3GalNAc-CGMC Cowper's gland mucin glycoprotein containing Gal3GalNAc side chains - MES 2-(N-morpholino) Ethane Sulfonic acid - PBS Phosphate Buffered Saline     

Heterogeneity of calcium compartmentation: Electron probe analysis of renal tubules

Summary The objective of this study has been to determine the intracellular localization of calcium in cryofixed, cryosectioned suspensions of kidney proximal tubules using quantitative electron probe X-ray microanalysis. Two populations of cells have been identified: 1) Viable cells, representing the majority of cells probed, are defined by their relatively normal K/Na concentration ratio of 41. Their measured Ca content is 4.1±1.4 (sem) mmol/kg dry wt in the cytoplasm and 3.1 ± 1.1 mmol/kg dry wt in the mitochondria, or an average cell calcium content of 3.8 mmol/kg dry wt. 2) Nonviable cells, defined by the presence of dense inclusions in their mitochondria and a K/Na concentration ratio of 1. The Ca content is 15±2 mmol/kg dry wt in the cytoplasm and 685±139 mmol/kg dry wt in the mitochondria of such cells. Assuming 25 to 30% of the cell volume is mitochondrial, the overall calcium content of such nonviable cells is 210 mmol/kg dry wt. The presence of these inclusions in 4 to 5% of the cells would account for the average total Ca content measured in perchloric acid extracts of isolated proximal tubule suspensions ( 18 nmol/mg protein or 12.6 mmol/kg dry wt). Whole kidney tissues display a large variability in toal Ca content (4.5 to 18 nmol/mg protein, or 3.4 to 13.5 mmol/kg dry wt), which could be accounted for by inclusion in 0 to 4% of the cells. The electron probe X-ray microanalysis (EPXMA) data conclusively demonstrate that thein situ mitochondrial Ca content of viable cells from the kidney, proximal tubule is low and support the idea that mitochondrial Ca may regulate dehydrogenase activity but probably does not normally control cytosolic free Ca.     

On the sufficiency of the velocity field for perception of heading

All models of self-motion from optical flow assume the instantaneous velocity field as input. We tested this assumption for human observers using random-dot displays that simulated translational and circular paths of movement by manipulating the lifetime and displacement of individual dots. For translational movement, observers were equally accurate in judging direction of heading from a velocity field with a two-frame dot life and a direction field in which the magnitudes of displacement were randomized while the radial pattern of directions was preserved, but at chance with a speed field in which the directions were randomized, preserving only magnitude. Accuracy declined with increasing noise in vector directions, but remained below 2.6° with a 90° noise envelope. Thus, the visual system uses the radial morphology of vector directions to determine translational heading and can tolerate large amounts of noise in this pattern. For circular movement, observers were equally accurate with a 2-frame velocity field, 3-frame acceleration displays, and 2-frame and 3-frame direction fields, consistent with the use of the pattern of vector directions to locate the center of rotation. The results indicate that successive independent velocity fields are sufficient for perception of translational and circular heading.     

13Cα and 13Cβ chemical shifts as a tool to delineate β-hairpin structures in peptides

Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of ¹³C and ¹³C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of ¹³C and ¹³C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of ¹³C and ¹³C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides.     

A structural model for elongation factor 1 (EF-1) and phosphorylation by protein kinase CKII

EF-1a binds aminoacyl-tRNA to the ribosome with the hydrolysis of GTP; the complex facilitates the exchange of GDP for GTP to initiate another round of elongation. To examine the subunit structure of EF-1 and phosphorylation by protein kinase CKII, recombinant , , and subunits from rabbit were expressed in E. coli and the subunits were reconstituted into partial and complete complexes and analyzed by gel filtration. To determine the availability of the and subunits for phosphorylation by CKII, the subunits and the reconstituted complexes were examined as substrates for CKII. Formation of the nucleotide exchange complex increased the rate of phosphorylation of the subunit and reduced the Km, while addition of to or the complex inhibited phosphorylation by CKII. However, a had little effect on phosphorylation of . Thus, the and subunits in EF-1 were differentially phosphorylated by CKII, in that phosphorylation of was altered by association with other subunits, while the site on was always available for phosphorylation by CKII. From the availability of the subunits for phosphorylation by CKII and the composition of the reconstituted partial and complete complexes, a model for the subunit structure of EF-1 consisting of (22)2 is proposed and discussed.     

Quantitative distribution of lysosomal hydrolases in the rat nephron

Summary The activities of N-acetyl-,d-glucosaminidase (NAG, EC 3.2.1.30),,d-galactosidase (-gal, EC 3.2.1.23) and acid phosphatase (ac-Pase, EC 3.1.3.2) were measured in the glomeruli, five segments of the proximal and four segments of the distal tubule of normal male Wistar rats. The activities of NAG and-gal are 3- to 5-fold higher in the first part of the proximal tubule than in other segments and very low in glomeruli. We propose that the distribution of these two glycosidases reflects the contribution of the different tubular segments to the reabsorption of glycoproteins. The maximal activity of ac-Pase was found in the straight part of the proximal tubule. It was only 1.5-fold higher than in the distal tubule. Moreover, the activity in glomeruli is rather high. We conclude that ac-Pase is not primarily involved in the handling of reabsorbed molecules.This research was supported by the Swiss National Science Foundation, grant 3.904-077     

Effects of exercise training and diabetes on cardiac myosin heavy chain composition

This study determined whether the beneficial effects of exercise training on the diabetic heart previously observed are associated with alterations in ventricular myosin heavy chain (MHC) isoform composition. Diabetes was induced in rats by i.v. streptozotocin. Trained rats were run on a treadmill for 60 min/day, 27 m/min, 10% grade. After 10 wks, ventricular MHC isoenzyme protein composition was analyzed for MHC composition using gel electrophoresis. -MHC and -MHC mRNA were determined by Northern and slot blot hybridization techniques. Both protein and mRNA analyses indicated that sedentary control rats exhibited a predominance of -MHC. Sedentary diabetics exhibited a shift to -MHC. Exercise trained diabetic rats showed a predominance of -MHC. The results indicate that treadmill exercise training of diabetic rat does not prevent the diabetes-induced shift in MHC composition towards the -MHC isoform, thus it is unlikely that the beneficial effects of exercise training on the diabetic heart, previously shown, are due to a normalization of the myosin isoform composition.     

Utilization of 5-fluorouracil byMyobacterium avium

Myobacterium avium LM1 was exposed to concentrations of 5-fluorouracil (5FU) that ranged from 0 to 100 g/ml. Growth inhibition was inversely proportional to the concentration of the drug. DNA was extracted from cells grown in medium that contained [¹⁴C]5FU, but no carrier. The [¹⁴C]DNA was enzymatically hydrolyzed to deoxyribonucleotides, which were separated and fractionated by high-performance liquid chromatography (HPLC). The isotope was located in 2-deoxycytidine 5-monophosphate (dCMP) and 2-deoxythymidine 5-monophosphate (dTMP), with dCMP containing the majority. There was no radioactivity at the elution times for 5-fluoro-2-deoxyuridine 5-monophosphate or 2-deoxyuridine 5-monophosphate. These results suggested that 5FU was dehalogenated and the uracil moiety ultimately converted into cytosine and thymine deoxyribonucleotides. Cells were grown in [³H]uracil, and [³H]DNA was extracted and analyzed by HPLC. The isotope was found only in the pyrimidine deoxyribonucleotides, with dCMP containing 4.1 times that in dTMP. Thus, it was demonstrated that uracil and dehalogenated 5FU were not directly incorporated into DNA, but rather converted to cytosine and thymine and then incorporated into DNA by a salvage pathway.     

Stimulation of non-specific resistance to tumors in the mouse using a poly(maleic-acid-styrene)-conjugated neocarzinostatin

Summary The development of non-specific resistance to tumors following stimulation with poly(maleic-acid-styrene)-conjugated neocarzinostatin (SMANCS), a polymer-conjugated derivative of neocarzinostatin, was investigated in mice. The growth of syngeneic solid tumors (Meth-A fibrosarcoma and RL 1 leukemia) inoculated into BALB/c mice was suppressed after one treatment with SMANCS at doses ranging from 0.14 mg/kg to 3.4 mg/kg i.v. 24 h before tumor implantation. Since previously observations concerning SMANCS have shown that it disappeared within 1.5 h after i.v. administration in mice and that it was inactivated quickly in plasma, SMANCS evidently inhibited tumor growth by mediating non-specific resistance. In addition, the non-specific resistance to tumors stimulated by SMANCS could be passively transferred to untreated mice by serum which was shown to contain interferon (IFN) from 12 h to 20 h after SMANCS administration. However, the resistance was not produced by serum prepared from mice at 8 h or 32 h after administration presumably because of the observation that the interferon activity was only demonstrated from 12 h to 28 h after SMANCS stimulation. When the serum specimens were treated with anti-IFN- antiserum, the antitumor activity of the sera was abrogated. However, no significant change was detected in the antitumor activity of the specimens following treatment with anti-IFN-/ antiserum. Treatment of mice with SMANCS and anti-IFN- antiserum together resulted in the elimination of the non-specific resistance to tumors. The IFN induced in the sera of mice by SMANCS was shown to be 57% IFN- and 41% IFN-/. Half of the interferon produced in SMANCS-stimulated mice could be eliminated by treatment with anti-IFN-, and treatment of SMANCS-stimulated mice with both anti-IFN- and anti-IFN-/ antisera resulted in a total absence of detectable interferon. These findings suggest that while the administration of SMANCS induces both IFN- and IFN-/ production, in this case, it is only the former which mediates the non-specific resistance to tumors.     

The effect of multiplicity of infection on recombination values in bacteriophageT 4 D

Zusammenfassung In Massenlysaten und Einzelwürfen vonT 4-Kreuzungen steigt der Prozentsatz der Rekombinanten mit steigender Infektionsmultiplizität an, und zwar sowohl zwischen gekoppelten als auch zwischen ungekoppelten Genen. Dieser Befund stimmt mitTrautners (1960) Ergebnissen beiT 1-Kreuzungen überein. Der scheinbarte Widerspruch zuEpsteins (1958) früheren Resultaten beiT 4-Kreuzungen läßt sich erklären, da verschiedene Wirtsbakterien verwendet wurden.Es wird diskutiert, ob dieser Effekt durch die Annahme erklärt werden kann, daß die infizierenden Phagegenome bei der Rekombination brechen und daß die Bruchstellen zufallsgemäß über die Genome einer Phagenpopulation verteilt sind. Die beschriebenen Versuche schließen jedochTrautners Interpretation nicht aus, daß in der infizierten Bakterienzelle eine Topographie existiert, d. h., daß die verschiedenen Genotypen während der Vermehrung nach Einzelinfektion nicht so vollständig durchmischt werden wie nach Mehrfachinfektion.

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With 2 Figures in the Text     

Callus proliferation from the protoplasts of embryogenic cells of Quercus serrata

Embryogenic culture was induced from the immature embryos of Quercus serrata using Marashige and Skoog's medium (MS) containing 0.1 M each of 2,4-d and BAP, and subcultured for seven months before isolation of protoplasts by using 1% Cellulase RS in 0.6 M mannitol solution. Efficient colony formation was obtained when protoplasts were cultured in a liquid MS medium containing 0.6 M mannitol, 3% sucrose and combination of 0.1 M or 1 M each of 2,4-d and BAP. Excluding ammonium nitrate from the MS medium resulted in the decrease of the percentage of colony formation. From colonies, both agar culture and liquid culture were sustained in the MS media without mannitol containing no plant growth regulator, or containing 0.1 M of BAP in combination with 0.1 M or 1 M of 2,4-d.Abbreviations BAP 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - MS medium after Murashige & Skoog (1962).     

Expression of the plant sulphite reductase in cell suspension cultures from Catharanthus roseus L.

Sulphur-heterotrophic growth exhibited a dual response to the expression of sulphate-assimilating enzymes. The level of ATP-sulphurylase (EC 2.7.7.4) appeared repressed while sulphite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulphhydrylase (EC 4.2.99.8) were derepressed and coordinated in their occurrence. The capability of the cells to reduce adenylylphosphosulphate or 3-phospho adenylylphosphosulphate to cysteine coincided with the activity of sulphite reductase. The expression of these reducing steps lacked correlation with the regulation of ATP-sulphurylase.Abbreviations APS adenylylphosphosulphate - MVH reduced methylviologen - OAS O-acetyl-l-serine - PAPS 3-phospho adenylylphosulphate     

“Hidden” popular illnesses in primary care: Residents' recognition and clinical implications

This study documents that ethnomedical beliefs and practices play an important role in primary care in a southern community. Thirty-three of 73 patients from a rural Appalachian area coming to a university primary care internal medicine practice presented 54 ethnomedical complaints such as high blood (24.1%), Weak 'n dizzy (22.2%), nerves (16.7%), sugar (5.6%) and fallin' out (3.7%). Thirty-three patients had both biomedical and ethnomedical complaints, 40 patients had biomedical complaints without ethnomedical complaints and no patients presented with ethnomedical complaints alone. Over two-thirds of all patients consulted non-medical personnel for their complaints, mostly family and friends, and 70 percent self-treated prior to clinic consultation. Patients presenting with ethnomedical complaints when compared with those presenting with biomedical complaints sought advice of non-physicians significantly more often (p < 0.02); no statistical difference, however, was found in their self-treatment practices. Ninety-two of 130 biomedical complaints were recorded by the patient's physician but none of the 54 ethnomedical complaints were formally recorded (p < 0.001). The high incidence of ethnomedical complaints in this population and the failure of physicians to recognize these complaints demand that primary care medicine residents be taught improved history-taking skills and the essentials of ethnomedical illnesses if they are to provide culturally-sensitive patient care. [ethnomedicine; physician education; Southern black and white Appalachian folk medicine; culturally-appropriate primary care.]     

Gibberellins play a role in the interaction between imidazole fungicides and cytokinins in Araceae

Imidazole fungicides such as imazalil, prochloraz, and triflurnizole and the triazole growth retardant paclobutrazol promote the shoot-inducing effect of exogenous cytokinins in Araceae, such as Spathiphyllum floribundum Schott and Anthurium andreanum Schott. The mechanism of their action could partially be based on the inhibition of gibberellic acid (GA) biosynthesis, because administration of GA₃ inhibits the phenomenon completely in S. floribundum. Not only is the suppression of GA biosynthesis involved, but also the metabolism of endogenous cytokinins is significantly altered. Although the balance between isopentenyladenine, zeatin, dihydrozeatin, and their derivatives was shifted to distinguished directions by administration of BA and/or imazalil and/or GA₃, no correlation between these changes in metabolic pathways and the number of shoots could be found. The metabolism of BA was not significantly altered by adding imazalil to the micropropagation medium of S. floribundum.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - [9R-5P]DHZ 9--d-ribofuranosyl-dihydrozeatin-monophosphate - [9R-5P]iP 6-isopentenyl-9--d-ribofuranosyladenine-monophosphate - [9R-5P]Z 9--d-ribofuranosyl-zeatin-monophosphate - [9G]BA 6-benzyl-9--d-glucopyranosyladenine - [9G]DHZ 9--d-glucopyranosyl-dihydrozeatin - [9G]iP 6-isopentenyl-9--d-glucopyranosyladenine - [9G]Z 9--d-glucopyranosyl-zeatin - [9R]BA 6-benzyl-9--d-ribofuranosyladenine - [9R]DHZ 9--d-ribofuranosyl-dihydrozeatin - [9R]iP 6-isopentenyl-9--d-ribofuranosyladenine - [9R]Z 9--d-ribofuranosyl-zeatin - BA 6-benzyladenine - DHZ dihydrozeatin - ES⁺ LC-MS/MS HPLC coupled Electrospray Tandem Mass Spectrometry - f.m. fresh mass - mT 6-(3-hydroxybenzyl)adenine - IMA imazalil - iP isopentenyladenine - NAA 1-naphthalene acetic acid - NFT Nutrient Film Technique - (OG)[9R]DHZ O--glucopyranosyl-9--d-ribofuranosyl-dihydrozeatin - (OG)[9R]Z O--d-glucopyranosyl-9--d-ribofuranosyl-zeatin - (OG)DHZ O--d-glucopyranosyl-dihydrozeatin - (OG)Z O--d-glucopyranosyl-zeatin - PAR Photosynthetic Active Radiation - PBZ paclobutrazol - PRO prochloraz - TDZ thidiazuron - TRI triflurnizole - Z zeatin     

Purification and properties of recombinant β-glucosidase of the hyperthermophilic bacterium Thermotoga maritima

A -glucosidase of the hyperthermophilic bacterium Thermotoga maritima has been purified from a recombinant Escherichia coli clone expressing the corresponding gene. The enzyme was found to be a dimer with an apparent molecular mass of approximately 95 kDa as determined by size exclusion chromatography. It was composed of two apparently identical subunits of about 47 kDa (determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis). The enzyme had a bbroadsubstrate specificity and attacked -glucoside, -galactoside, -fucoside, and, to a very small extent, also -xyloside substrates. -Glycosidic bonds were not hydrolysed. Kinetic measurement of the hydrolysis of o-nitrophenyl--d-glucopyranoside (oNPGlc) and o-nitrophenyl--d-galactopyranoside (oNPGal) in the concentration ranges 0.05–20 mm and 0.1–10 mm, respectively, at 75°C resulted in non-linear Lineweaver-Burk and Eadie-Hofstee 3lots whereas cellobiose and lactose did not induce this type of effect. Lactose caused substrate inhibition above 350 mm. The enzyme was optimally active at about pH 6.1. The T. maritima -glucosidase represents the most thermostable -glucosidase described to date. In 50 mm sodium phosphate buffer, pH 6.2, at an enzyme concentration of 50 g/ml, the pure enzyme without additives retained more than 60% of its initial activity after a 6-h incubation at 95°C. Correspondence to: W. Liebl